WO2012020759A1 - Transcriptase inverse variante - Google Patents

Transcriptase inverse variante Download PDF

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Publication number
WO2012020759A1
WO2012020759A1 PCT/JP2011/068157 JP2011068157W WO2012020759A1 WO 2012020759 A1 WO2012020759 A1 WO 2012020759A1 JP 2011068157 W JP2011068157 W JP 2011068157W WO 2012020759 A1 WO2012020759 A1 WO 2012020759A1
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residue
amino acid
reverse transcriptase
seq
substitution
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PCT/JP2011/068157
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Japanese (ja)
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保川　清
國世 井上
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国立大学法人京都大学
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Priority to US13/816,497 priority Critical patent/US8900814B2/en
Priority to JP2012528684A priority patent/JPWO2012020759A1/ja
Priority to EP11816423.5A priority patent/EP2604688B1/fr
Publication of WO2012020759A1 publication Critical patent/WO2012020759A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Definitions

  • the present invention relates to a mutant reverse transcriptase. More specifically, the present invention relates to a mutant reverse transcriptase, a nucleic acid encoding the same, a reverse transcription method using the mutant reverse transcriptase, the mutant reverse transcriptase, which are useful for gene analysis, disease inspection, and the like. And a method for improving the thermal stability of nucleic acid-related enzymes such as the mutant reverse transcriptase.
  • Reverse transcriptase generally has an activity of synthesizing cDNA using RNA as a template (hereinafter referred to as “RNA-dependent DNA polymerase activity”) and an activity of synthesizing DNA using DNA as a template (hereinafter referred to as “DNA-dependent DNA”). And the activity of degrading the RNA strand in the RNA: DNA hybrid (hereinafter referred to as “RNase H activity”).
  • reverse transcriptase Since such reverse transcriptase has the RNA-dependent DNA polymerase activity, for example, analysis of the base sequence of mRNA that directly reflects the amino acid sequence of the protein expressed in the living body, cDNA library It is used for construction and RT-PCR. Conventionally, Moloney murine leukemia virus reverse transcriptase or avian myeloblastosis virus reverse transcriptase is used for such applications.
  • the present invention has been made in view of the above prior art, and an object thereof is to provide a mutant reverse transcriptase having high thermostability and high versatility. Another object of the present invention is to provide a nucleic acid and a method for producing the mutant reverse transcriptase from which the mutant reverse transcriptase can be easily obtained. Another object of the present invention is to provide a versatile reverse transcription reaction kit and detection kit. Furthermore, an object of the present invention is to provide a method for improving the thermal stability of a nucleic acid-related enzyme that can greatly improve the thermal stability of the nucleic acid-related enzyme. It is another object of the present invention to provide a reverse transcription method that is highly versatile and can perform a reverse transcription reaction efficiently.
  • the gist of the present invention is as follows.
  • a mutant reverse transcriptase, characterized in that it has a DNA interaction region having a larger positive net charge and exhibits reverse transcriptase activity [2]
  • the wild type reverse transcriptase consists of an amino acid sequence corresponding to SEQ ID NO: 2,
  • the amino acid residue in the DNA interaction region of the wild type reverse transcriptase is an amino acid residue localized in the region corresponding to the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2.
  • the amino acid residue has a conservative substitution of amino acid residues in the region corresponding to the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2.
  • the mutant reverse transcriptase according to [2] or [3] above [5] At least one of the negatively charged amino acid residues among the amino acid residues localized in the region corresponding to the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2 is a positively charged amino acid residue.
  • an amino acid residue corresponding to the glutamic acid residue at position 286 of SEQ ID NO: 2 is substituted with a positively charged amino acid residue or a nonpolar amino acid residue , And exhibiting reverse transcriptase activity, a mutant reverse transcriptase, [7]
  • the amino acid residue in SEQ ID NO: 2 Glutamic acid residue at position 69, Aspartic acid residue at position 108, Glutamic acid residue at position 117, Aspartic acid residue at position 124, Glutamic acid residue at position 286, Glutamic acid residue at position 302, Tryptophan residue at position 313
  • a residue corresponding to at least one of a leucine residue at position 435 and an asparagine residue at position 454 is substituted with a positively charged amino acid
  • a mutant reverse transcription comprising an amino acid sequence having at least one selected from the group consisting of substitution of a residue corresponding to alanine residue or arginine residue, and exhibiting reverse transcriptase activity enzyme, [9] (I) In the amino acid sequence corresponding to SEQ ID NO: 2, the following amino acid residue substitutions (a-1) to (c-1): (A-1) substitution of the residue corresponding to the glutamic acid residue at position 286 of SEQ ID NO: 2 with an alanine residue; (B-1) substitution of a residue corresponding to the glutamic acid residue at position 302 of SEQ ID NO: 2 with a lysine residue, and (c-1) a residue corresponding to a leucine residue at position 435 of SEQ ID NO: 2.
  • the mutant reverse transcriptase of the present invention has excellent properties such as high heat stability and high versatility. Further, according to the nucleic acid of the present invention and the method for producing the mutant reverse transcriptase of the present invention, the mutant reverse transcriptase can be easily obtained. Moreover, the reverse transcription reaction kit and the detection kit of the present invention have excellent properties such as high versatility. Furthermore, according to the method for improving the thermal stability of the nucleic acid-related enzyme of the present invention, the thermal stability of the nucleic acid-related enzyme can be greatly improved. Further, the reverse transcription method of the present invention is highly versatile, and according to the method, a reverse transcription reaction can be performed efficiently.
  • FIG. 6 is a drawing-substituting photograph showing the result of electrophoresis in Test Example 5.
  • the present inventors introduce positively charged amino acid residues or nonpolar amino acid residues into the DNA interaction region of the reverse transcriptase to make the positive net charge larger than the DNA interaction region of the reverse transcriptase before the introduction. Thus, it was found that the thermal stability of reverse transcriptase is greatly improved.
  • the present invention is based on such knowledge.
  • mutant reverse transcriptase In the mutant reverse transcriptase of the present invention, the amino acid residue in the DNA interaction region of wild type reverse transcriptase is substituted with a positively charged amino acid residue or a nonpolar amino acid residue, It has a DNA interaction region having a positive net charge larger than the DNA interaction region of the type reverse transcriptase, and exhibits reverse transcriptase activity.
  • the mutant reverse transcriptase of the present invention Since the mutant reverse transcriptase of the present invention has the DNA interaction region, it exhibits high thermal stability. Therefore, according to the mutant reverse transcriptase of the present invention, the reverse transcription reaction can be performed even at a high reaction temperature. Therefore, even when the RNA has a base sequence that tends to form a secondary structure, the reaction temperature is reduced. By increasing the height, cDNA can be synthesized while suppressing the formation of secondary structure. Therefore, according to the mutant reverse transcriptase, a highly versatile reverse transcription reaction can be performed.
  • wild-type reverse transcriptase refers to a reverse transcriptase in which no artificial mutation has been introduced (hereinafter also referred to as “WTRT”).
  • WTRT include reverse transcriptase having an amino acid sequence corresponding to SEQ ID NO: 2.
  • amino acid sequence corresponding to SEQ ID NO: 2 means the reverse transcription comprising the amino acid sequence shown in SEQ ID NO: 2 (Moloney murine leukemia virus reverse transcriptase) and the amino acid sequence shown in SEQ ID NO: 2.
  • An amino acid sequence of an enzyme ortholog eg, avian myeloblastosis virus reverse transcriptase, human immunodeficiency virus reverse transcriptase).
  • mutant reverse transcriptase refers to a reverse transcriptase into which a mutation has been artificially introduced.
  • MMLV reverse transcriptase “Moloney murine leukemia virus reverse transcriptase” may be referred to as “MMLV reverse transcriptase”.
  • DNA interaction region refers to a region where amino acid residues that cause an interaction with DNA are localized in reverse transcriptase.
  • “having a positive net charge larger than the DNA interaction region of wild-type reverse transcriptase” means a pH suitable for carrying out a reverse transcription reaction (eg, pH 6.0 to 9.5). ) Means larger than the positive net charge of the DNA interaction region of the wild type reverse transcriptase.
  • the magnitude of the net charge is, for example, “+1” for the charge scores of lysine residues and arginine residues, which are positively charged amino acid residues, and the charges of aspartic acid residues and glutamic acid residues, which are negatively charged amino acid residues.
  • the score is “ ⁇ 1”, based on the number of lysine residues, arginine residues, aspartic acid residues and glutamic acid residues in the DNA interaction region, the formula (I):
  • Effective charge magnitude score (+ 1 ⁇ k) + (+ 1 ⁇ r) + ( ⁇ 1 ⁇ d) + ( ⁇ 1 ⁇ e) (I)
  • k represents the number of lysine residues
  • r represents the number of arginine residues
  • d represents the number of aspartic acid residues
  • e represents the number of glutamic acid residues.
  • the effective charge magnitude score calculated using the above formula (I) is the effective charge in the DNA interaction region of the wild type MMLV reverse transcriptase. It is desirable that positively charged amino acid residues or nonpolar amino acid residues are localized so as to be larger than the score (+7).
  • the effective charge magnitude score of the DNA interaction region of the mutant reverse transcriptase of the present invention is usually +8 to +13, preferably +9, from the viewpoint of ensuring high thermal stability and ensuring high specific activity. To +13, more preferably +11 to +13.
  • the positively charged amino acid residue examples include an arginine residue, a lysine residue, and a histidine residue.
  • it is positively charged at a pH suitable for performing a reverse transcription reaction (for example, pH 6.0 to 9.5), and high thermal stability can be secured under such pH conditions.
  • a pH suitable for performing a reverse transcription reaction for example, pH 6.0 to 9.5
  • high thermal stability can be secured under such pH conditions.
  • an arginine residue and a lysine residue Preferably an arginine residue and a lysine residue.
  • nonpolar amino acid residues examples include alanine residues, glycine residues, valine residues, leucine residues, isoleucine residues, methionine residues, cysteine residues, tryptophan residues, phenylalanine residues, and proline residues. Etc. Among these, an alanine residue is preferable because the size of the side chain is small and the shape change caused by substitution is considered to be small.
  • the reverse transcriptase activity can be measured by performing the following steps (1) to (6).
  • Reaction solution [Composition: 25 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 2 mM dithiothreitol, 5 mM magnesium chloride, 12.5 ⁇ M poly (rA) ⁇ p (dT) 15 (p (dT) 15 in terms of concentration), and 0.2 mM [methyl - 3 H] dTTP] steps of incubation at 37 ° C.
  • step (2) collecting 20 ⁇ L of the product obtained in step (1) and spotting it on a glass filter; (3) The glass filter after the step (2) is washed with a cooled 5% by mass trichloroacetic acid aqueous solution for 10 minutes, and then washed with a cooled 95% by volume ethanol aqueous solution three times.
  • WTRT has an amino acid sequence corresponding to SEQ ID NO: 2, and amino acid residues in the WTRT DNA interaction region. Are preferably amino acid residues localized in a region corresponding to the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2 (hereinafter also referred to as “region T24-P474 ”).
  • region T24-P474 amino acid residues localized in a region corresponding to the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2
  • regions T24-P474 from the viewpoint of ensuring high thermal stability and ensuring sufficient specific activity, a region corresponding to the serine residue at position 60 to the glutamine residue at position 84 of SEQ ID NO: 2.
  • region S60-Q84 an asparagine residue at position 95 to a cysteine residue at position 157 (hereinafter also referred to as “region N95-C157 ”), a glutamine residue at position 190 A region corresponding to an asparagine residue at position 194 (hereinafter also referred to as “region Q190-N194 ”), a region corresponding to a leucine residue at position 220 to a glutamic acid residue at position 233 (hereinafter referred to as “region L220-E233 ”) A region corresponding to a lysine residue at position 257 to a glutamic acid residue at position 275 (hereinafter also referred to as “region K257-E275 ”); a region corresponding to a leucine residue at position 280 to a threonine residue at position 287; (hereinafter referred to as "area L280-T287" Also referred to), the region corresponding to the leu
  • the mutant reverse transcriptase of the present invention has a conservative substitution of an amino acid residue in the region T24-P474 in the amino acid sequence corresponding to SEQ ID NO: 2 within a range that does not interfere with the object of the present invention. Also good.
  • the conservative substitution include a specific amino acid residue and an amino acid residue that exerts a function similar to the specific amino acid residue in terms of hydrophobicity, charge, pKa, steric features, and the like. Substitution etc. are mentioned. More specifically, examples of the conservative substitution include substitution of one amino acid residue belonging to any of the following groups I to VI with another amino acid residue belonging to the same group.
  • Group I Glycine and alanine residues
  • Group II Valine, isoleucine and leucine residues
  • Group III Aspartate, glutamate, asparagine and glutamine residues
  • Group IV Serine and Threonine residue
  • Group V Lysine residue and arginine residue
  • Group VI Phenylalanine residue and tyrosine residue
  • mutant reverse transcriptase of the present invention is within a range that does not interfere with the object of the present invention.
  • A Substitution of one or several amino acid residues in the sequence excluding the region corresponding to the threonine residue at position 24 to the proline residue at position 474 in the amino acid sequence corresponding to SEQ ID NO: 2
  • An amino acid sequence further having a deletion, insertion or addition and
  • B a sequence excluding the region corresponding to the threonine residue at position 24 to the proline residue at position 474 of SEQ ID NO: 2, by the BLAST algorithm, It has any amino acid sequence of the amino acid sequence having sequence identity of at least 80% obtained by alignment under the conditions of Gap Costs (Existence 11, Extension 1), Expect 10, and Word Size 3. It may be an enzyme exhibiting a coenzyme activity.
  • substitution, deletion, insertion or addition of one or several amino acid residues means substitution, deletion, insertion or addition of a number of amino acid residues within a range that gives a polypeptide exhibiting reverse transcriptase activity. To do. “One or several” specifically refers to 1 to 30, preferably 1 to 20, more preferably 1 to 10, and more preferably 1 to 3.
  • sequence identity is aligned under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3 by the BLAST algorithm from the viewpoint of ensuring high thermal stability and sufficient specific activity.
  • the calculated value is 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100%.
  • the mutant reverse transcriptase of the present invention is characterized in that at least one of the negatively charged amino acid residues among the amino acid residues localized in the region T24-P474 is the positively charged amino acid. It is preferably substituted with a residue or a nonpolar amino acid residue.
  • Examples of the negatively charged amino acid residue include an aspartic acid residue and a glutamic acid residue.
  • Such a negatively charged amino acid residue is such that a polypeptide in which the negatively charged amino acid residue is substituted with the positively charged amino acid residue or the nonpolar amino acid residue in the WTRT amino acid sequence can express reverse transcriptase activity. In this case, it may be a residue present at a position that causes a change in shape.
  • the mutant reverse transcriptase of the present invention is the amino acid sequence corresponding to SEQ ID NO: 2 as the negatively charged amino acid residue from the viewpoint of ensuring high thermal stability and ensuring sufficient specific activity. :
  • the amino acid residue corresponding to the glutamic acid residue at position 286 of 2 is substituted with the positively charged amino acid residue or the nonpolar amino acid residue, and exhibits reverse transcriptase activity.
  • the mutant reverse transcriptase of the present invention is an amino acid residue other than the amino acid residue corresponding to the glutamic acid residue at position 286 among the amino acid residues localized in the region T24-P474 within the range that does not interfere with the object of the present invention. Negatively charged amino acid residues and / or other amino acid residues may be substituted with the positively charged amino acid residues or nonpolar amino acid residues.
  • the mutant reverse transcriptase of the present invention is an amino acid residue corresponding to SEQ ID NO: 2, in which amino acid residues in SEQ ID NO: 2 are glutamic acid residues at position 69 and aspartic acid residues in position 108.
  • 117-position glutamic acid residue, 124-position aspartic acid residue, 286-position glutamic acid residue, 302-position glutamic acid residue, 313-position tryptophan residue, 435-position leucine residue, and 454-position asparagine residue The enzyme corresponding to at least one of these may be substituted with a positively charged amino acid residue or a nonpolar amino acid residue, and exhibit reverse transcriptase activity.
  • substitution of the glutamic acid residue at position 302 with arginine can improve the thermal stability as compared with WTRT, but from the viewpoint of ensuring higher thermal stability, the substitution of the glutamic acid residue at position 302 is Substitution for arginine is excluded.
  • the mutant reverse transcriptase of the present invention has the following amino acid residue substitutions (a) to (i) in the amino acid sequence corresponding to SEQ ID NO: 2 from the viewpoint of ensuring higher thermostability: (A) substitution of the residue corresponding to the glutamic acid residue at position 286 of SEQ ID NO: 2 with an alanine residue, a lysine residue or an arginine residue; (B) substitution of the residue corresponding to the glutamic acid residue at position 302 of SEQ ID NO: 2 with an alanine residue or a lysine residue; (C) substitution of the residue corresponding to the leucine residue at position 435 of SEQ ID NO: 2 with an alanine residue, lysine residue or arginine residue; (D) substitution of the residue corresponding to the aspartic acid residue at position 124 of SEQ ID NO: 2 with an alanine residue, a lysine residue or an arginine residue; (E) substitution of the residue corresponding to the gluta
  • the mutant reverse transcriptase of the present invention has one mutation selected from the amino acid residue substitutions (a) to (i) above (that is, when it is a single mutant), it has a higher heat.
  • (A-1) substitution of the residue corresponding to the glutamic acid residue at position 286 of SEQ ID NO: 2 with an alanine residue (B-1) substitution of a residue corresponding to the glutamic acid residue at position 302 of SEQ ID NO: 2 with a lysine residue, and (c-1) a residue corresponding to a leucine residue at position 435 of SEQ ID NO: 2.
  • amino acid sequence having a substitution of a group with an arginine residue and (d-1) an amino acid sequence having either a substitution of a residue corresponding to the aspartic acid residue at position 124 of SEQ ID NO: 2 with an arginine residue And preferably exhibits reverse transcriptase activity.
  • the mutant reverse transcriptase of the present invention is higher when it has a plurality of types of mutations selected from the amino acid residue substitutions (a) to (i) (that is, when it is a multiple mutant).
  • the following amino acid residue substitutions (a-1) to (c-1) (A-1) substitution of the residue corresponding to the glutamic acid residue at position 286 of SEQ ID NO: 2 with an alanine residue; (B-1) substitution of a residue corresponding to the glutamic acid residue at position 302 of SEQ ID NO: 2 with a lysine residue, and (c-1) a residue corresponding to a leucine residue at position 435 of SEQ ID NO: 2.
  • amino acid sequence having a substitution of a group with an arginine residue or (II) an arginine of a residue corresponding to the aspartic acid residue at position 124 of (d-1) SEQ ID NO: 2 in the amino acid sequence of (I) above It preferably consists of an amino acid sequence further having substitution to a residue and exhibits reverse transcriptase activity.
  • the mutant reverse transcriptase of the present invention is the (e-1) sequence in the amino acid sequence of (I) or (II). It may be any amino acid sequence that further has substitution of the residue corresponding to the aspartic acid residue at position 524 of No. 2 with an alanine residue and that exhibits reverse transcriptase activity.
  • nucleic acid encoding mutant reverse transcriptase The nucleic acid of the present invention is a nucleic acid encoding the mutant reverse transcriptase of the present invention. Since the nucleic acid of the present invention encodes the mutant reverse transcriptase, the mutant reverse transcriptase can be easily obtained by expressing the mutant reverse transcriptase encoded by the nucleic acid.
  • nucleic acid examples include DNA and mRNA, but the present invention is not limited to such examples.
  • the nucleic acid of the present invention is, for example, a site-specific mutation relative to a nucleic acid encoding WTRT so that an amino acid residue in the WTRT DNA interaction region is replaced with a positively charged amino acid residue or a nonpolar amino acid residue. Can be obtained.
  • the mutant reverse transcriptase of the present invention can be obtained by expressing the mutant reverse transcriptase encoded by the nucleic acid using the nucleic acid of the present invention.
  • the present invention also includes a method for producing such a mutant reverse transcriptase.
  • the production method of the present invention is a method for producing the aforementioned mutant reverse transcriptase, (I) a step of culturing cells retaining the nucleic acid of the present invention to express a mutant reverse transcriptase encoded by the nucleic acid to obtain a culture, and (ii) a mutation from the culture obtained in the step
  • the method includes a step of recovering the type reverse transcriptase.
  • cells retaining the nucleic acid of the present invention are cultured to express a mutant reverse transcriptase encoded by the nucleic acid to obtain a culture [“step (i)”].
  • the cell retaining the nucleic acid can be obtained, for example, by transforming a host cell using a carrier for gene introduction containing the nucleic acid.
  • Examples of the host cell include bacterial cells such as E. coli, insect cells, yeast cells, plant cells, and animal cells, but the present invention is not limited to such examples. Among these, a mutant reverse transcriptase can be easily purified, and a large amount of mutant reverse transcriptase can be produced. Therefore, a bacterial cell is preferable, and an E. coli cell is more preferable. Examples of the E. coli cells include BL21 (DE3), but the present invention is not limited to such examples.
  • the carrier for gene introduction may be a biological carrier or a non-biological carrier.
  • biological carriers include vectors such as plasmid vectors, phage vectors, and viral vectors, but the present invention is not limited to such examples.
  • non-biological carrier include gold particles, dextran, and liposomes, but the present invention is not limited to such examples.
  • Such a carrier for gene transfer can be appropriately selected depending on the host cell to be used. For example, when the host cell is Escherichia coli, a plasmid vector or a phage vector can be used as a carrier for gene introduction.
  • the host cell is BL21 (DE3) which is Escherichia coli, a pET plasmid vector can be used. In this case, the mutant reverse transcriptase can be expressed in a large amount, and the mutant reverse transcriptase can be easily purified.
  • the vector may contain elements for facilitating purification of the mutant reverse transcriptase, such as an extracellular secretion signal, a His tag, and the like.
  • the gene introduction carrier When the gene introduction carrier is a plasmid vector, phage vector or virus vector which is the biological carrier, the gene introduction carrier inserts the nucleic acid into a cloning site of a plasmid vector, phage vector or virus vector, and a promoter. It can be produced by operably connecting under the control of
  • “operably linked” means that the expression of a polypeptide encoded by a nucleic acid is linked so that it is expressed in a state exhibiting biological activity under the control of an element such as a promoter.
  • the carrier for gene introduction is the non-biological carrier
  • the carrier for gene introduction is a nucleic acid construct obtained by operably linking the nucleic acid under the control of a promoter, if necessary. , And can be prepared by supporting the non-biological carrier.
  • a nucleic acid construct may appropriately contain elements necessary for expression of a gene such as a replication origin and a terminator.
  • Transformation can be performed by a transformation method according to the type of gene introduction carrier used.
  • transformation methods include an electroporation method, a calcium phosphate method, a DEAE-dextran method, and a particle gun method, but the present invention is not limited to such examples.
  • the culture conditions of the cells holding the nucleic acid can be appropriately set according to the type of host cell used.
  • the nucleic acid is operably linked under the control of an inducible promoter
  • cells that retain the nucleic acid may be cultured under expression-inducing conditions according to the type of the promoter.
  • the mutant reverse transcriptase is recovered from the culture obtained in the step (i) [“step (ii)”].
  • the culture is subjected to centrifugation or the like to recover the cells, and the mutant reverse transcriptase may be isolated from the cells.
  • the cells are disrupted by an ultrasonic disruption method, a lysis method, a freeze disruption method, etc., and the resulting cell extract is centrifuged, ultracentrifuged, ultrafiltration, salting out, dialysis, ion exchange column
  • the mutant reverse transcriptase can be isolated by subjecting it to chromatography, adsorption column chromatography, affinity chromatography, gel filtration column chromatography and the like.
  • the mutant reverse transcriptase when secreted outside the cell, the culture is subjected to centrifugation, filtration or the like, and the culture supernatant is collected, and the mutant reverse transcriptase may be isolated from the culture supernatant.
  • the reverse transcription method of the present invention is characterized by synthesizing cDNA from RNA using the mutant reverse transcriptase of the present invention.
  • the mutant reverse transcriptase of the present invention has a higher thermal stability than the wild type reverse transcriptase. Therefore, according to the reverse transcription method of the present invention, the reverse transcription reaction can be performed in a wide temperature range including a high temperature sufficient to suppress the formation of RNA secondary structure. Therefore, the reverse transcription method of the present invention can perform a reverse transcription reaction efficiently regardless of the type of RNA, and is highly versatile.
  • the mutant reverse transcriptase, RNA, an oligonucleotide primer complementary to the RNA, and four types of deoxyribonucleoside triphosphates are incubated in a reverse transcription reaction buffer.
  • a reverse transcription reaction can be performed.
  • the reaction temperature in the reverse transcription reaction varies depending on the type of RNA used, the type of mutant reverse transcriptase used, etc., and therefore is appropriately set according to the type of RNA used, the type of mutant reverse transcriptase used, etc. It is preferable.
  • the reaction temperature can be set to 37 to 45 ° C., for example, when the RNA used is an RNA that is difficult to form a secondary structure at a reaction temperature suitable for WT.
  • the reaction temperature is, for example, a temperature higher than the reaction temperature suitable for WT, for example, 45 to 60 ° C. when the RNA used is an RNA that easily forms a secondary structure at a reaction temperature suitable for WT.
  • the reverse transcriptase concentration in the reaction system during the reverse transcription reaction varies depending on the use of the reverse transcription method of the present invention, it is preferably set as appropriate according to the use.
  • the concentration of the reverse transcriptase is usually preferably 0.001 to 0.1 ⁇ M.
  • the concentration of the oligonucleotide primer in the reaction system during the reverse transcription reaction is usually preferably 0.1 to 10 ⁇ M.
  • concentrations of the four types of deoxyribonucleoside triphosphates in the reaction system during the reverse transcription reaction differ depending on the concentration and length of the target RNA, the concentration depends on the concentration and length of the target RNA. It is preferable to set appropriately.
  • concentration of the four deoxyribonucleoside triphosphates is usually preferably 0.01 to 1 ⁇ M.
  • the reverse transcription reaction buffer can be appropriately selected according to the type of mutant reverse transcriptase used.
  • the reverse transcription reaction buffer may contain a divalent cation, such as magnesium ion or manganese ion.
  • the reverse transcription reaction buffer is not limited to the purpose of the present invention, and if necessary, a reducing agent (for example, dithiothreitol), a stabilizer (for example, glycerol, trehalose, etc.), organic Components such as a solvent (for example, dimethyl sulfoxide, formamide, etc.) may be contained.
  • the concentration of the divalent cation in the reverse transcription reaction buffer solution varies depending on the type of reverse transcriptase and other components contained in the reverse transcription reaction buffer solution. It is preferable to set appropriately according to other components contained in the buffer solution for the photoreaction.
  • the concentration of the divalent cation is usually 1 to 30 mM.
  • the pH of the reverse transcription reaction buffer varies depending on the type of reverse transcriptase and other components contained in the reverse transcription reaction buffer, it is included in the type of reverse transcriptase and the reverse transcription reaction buffer. It is preferable to set appropriately according to other components.
  • the pH of the reverse transcription reaction buffer is generally 6.0 to 9.5.
  • the reverse transcription reaction kit of the present invention is a kit for carrying out a reverse transcription reaction, and is characterized by containing the mutant reverse transcriptase of the present invention. Since the reverse transcription reaction kit of the present invention contains the mutant reverse transcriptase of the present invention having high thermal stability, a wide temperature range including a high temperature sufficient to suppress the formation of RNA secondary structure. Suitable for reverse transcription reaction in a range. Therefore, the reverse transcription reaction kit of the present invention is highly versatile because the reverse transcription reaction can be efficiently performed regardless of the type of RNA.
  • the reverse transcription reaction kit of the present invention may contain a reagent necessary for performing a reverse transcription reaction in addition to the mutant reverse transcriptase.
  • a reagent necessary for performing a reverse transcription reaction in addition to the mutant reverse transcriptase.
  • examples of such a reagent include RNA used as a template for a reverse transcription reaction, oligonucleotide primers complementary to the RNA, four types of deoxyribonucleoside triphosphates, a buffer for reverse transcription reaction, an organic solvent, and the like.
  • the reverse transcription reaction buffer is the same as the reverse transcription reaction buffer used in the reverse transcription method.
  • the mutant reverse transcriptase may be enclosed in a container containing a storage buffer containing a stabilizer such as glycerol or trehalose.
  • a storage buffer include a buffer having a pH corresponding to the pH stability of the mutant reverse transcriptase.
  • the reagent necessary for performing the reverse transcription reaction may be enclosed in a container different from the container containing the mutant reverse transcriptase, and the progress of the reverse transcription reaction during storage of the reagent. May be enclosed in the same container as the mutant reverse transcriptase.
  • the reagent may be enclosed in a container so as to have an amount suitable for performing a reverse transcription reaction. This eliminates the need for the user to mix each reagent in an amount suitable for the reverse transcription reaction, and is easy to handle.
  • the detection kit of the present invention is a kit for detecting a marker in a sample containing RNA obtained from a living body, and contains the mutant reverse transcriptase and the reagent for detecting the marker. It is a feature. Since the detection kit of the present invention contains the mutant reverse transcriptase having high thermostability, reversal over a wide temperature range including a high temperature sufficient to suppress the formation of RNA secondary structure. Suitable for photoreaction. Therefore, the detection kit of the present invention can be used for various samples and is highly versatile.
  • the marker examples include RNA having a base sequence peculiar to viruses or bacteria contained in a living body, a base sequence peculiar to a disease, and the like.
  • the “base sequence peculiar to a virus or a bacterium” refers to a base sequence that exists in a virus or bacterium but does not exist in a living body.
  • the “base sequence peculiar to a disease” refers to a base sequence that exists in a living body affected with a disease but does not exist in a normal living body that does not suffer from the disease.
  • the virus is not particularly limited, and examples thereof include HPV, HIV, influenza virus, HCV, Norovirus, West Nile virus and the like.
  • examples of the bacterium include Bacillus cereus, Salmonella, enterohemorrhagic Escherichia coli, Vibrio, Campylobacter, and methicillin-resistant Staphylococcus aureus that cause food poisoning.
  • examples of the disease include cancer, diabetes, heart disease, high blood pressure, and infectious diseases.
  • the reagent for detecting the marker examples include a probe that is complementary to the RNA serving as the marker and bound with a fluorescent substance or a radioactive substance, or a fluorescent substance that specifically intercalates with a double-stranded nucleic acid (for example, Ethidium bromide).
  • the detection kit of the present invention includes, for example, four types of deoxyribonucleoside triphosphates, a reverse transcription reaction buffer, an organic solvent, RNA serving as a positive standard, It may contain RNA that is a negative standard.
  • the reverse transcription reaction buffer is the same as the reverse transcription reaction buffer used in the reverse transcription method.
  • the mutant reverse transcriptase may be enclosed in a container containing a storage buffer containing a stabilizer such as glycerol or trehalose.
  • a storage buffer solution is the same as the storage buffer solution in the reverse transcription reaction kit.
  • the four types of deoxyribonucleoside triphosphates, reverse transcription reaction buffer, and the like may be sealed in a container different from the container containing the mutant reverse transcriptase, and the reagent may be stored. As long as the progress of the reverse transcription reaction therein is stopped, it may be enclosed in the same container as the mutant reverse transcriptase. From the same viewpoint as in the case of the reverse transcription reaction kit, the reagent may be enclosed in a container so as to have an amount suitable for performing the reverse transcription reaction.
  • the method for improving the thermal stability of a nucleic acid-related enzyme according to the present invention is a method for improving the thermal stability of a nucleic acid-related enzyme having a nucleic acid interaction region that interacts with a nucleic acid. , Replacing amino acid residues in the nucleic acid interaction region with positively charged amino acid residues or nonpolar amino acid residues with respect to the base sequence corresponding to the nucleic acid interaction region in the nucleic acid encoding the wild-type nucleic acid-related enzyme The mutation is introduced to form a nucleic acid interaction region having a positive net charge larger than the nucleic acid interaction region of the wild-type nucleic acid-related enzyme.
  • the nucleic acid-related enzyme has a nucleic acid interaction region that interacts with a nucleic acid. Therefore, as in the case of the mutant reverse transcriptase of the present invention, high thermal stability is ensured by substituting amino acid residues in the nucleic acid interaction region with positively charged amino acid residues or nonpolar amino acid residues. Expected to be able to.
  • the nucleic acid-related enzyme may be an enzyme having a nucleic acid interaction region that interacts with a nucleic acid.
  • examples of the nucleic acid-related enzyme include reverse transcriptase, DNA polymerase, restriction enzyme, methylase, RNA polymerase, and telomerase. Of these, reverse transcriptase is preferable because thermal stability can be further improved.
  • the introduction of the mutation into the nucleotide sequence corresponding to the nucleic acid interaction region in the nucleic acid encoding the wild type nucleic acid-related enzyme is such that the amino acid residues in the nucleic acid interaction region of the wild type nucleic acid related enzyme are positively charged amino acid residues or non- PCR can be performed using primers designed to be substituted with polar amino acid residues.
  • the position of the amino acid residue to be mutated differs depending on the type of the nucleic acid-related enzyme, but in the nucleic acid interaction region of the nucleic acid-related enzyme, the position of the nucleic acid-related enzyme is close to the nucleic acid phosphate group. Positions of amino acid residues that do not cause a change in shape that hinders activity, positions of amino acid residues that are close to the negatively charged amino acid residue of reverse transcriptase and do not cause a change in shape that hinders the activity of nucleic acid-related enzymes, etc. Is mentioned.
  • a nucleic acid-related enzyme with improved thermal stability can be produced by using a nucleic acid into which a mutation has been introduced, in the same manner as the method for producing a mutant reverse transcriptase.
  • Escherichia coli BL21 (DE3) was transformed with the obtained plasmid.
  • the obtained cells were cultured at 30 ° C. in L broth containing 50 ⁇ g / mL ampicillin to obtain transformed cells.
  • the transformed cells were inoculated into 3 mL of L broth containing 50 ⁇ g / mL ampicillin and incubated at 30 ° C. for 16 hours with shaking. Thereafter, the transformed cells were cultured in an automatic induction system (manufactured by Novagen, trade name: Overnight Express Automation System) to express the protein.
  • an automatic induction system manufactured by Novagen, trade name: Overnight Express Automation System
  • the resulting culture was subjected to a cell lysis reagent (Promega), product name: FastBreak Cell Lysation Reagent, which is included in a protein purification system (Promega, product name: HisLink Spin Protein Purification System). ] was added to lyse the transformed cells.
  • a resin for protein purification (trade name: HisLink Protein Purification Resin, manufactured by Promega) included in the protein purification system was added to the obtained lysate.
  • the lysate containing the resin was transferred to a column [Promega, trade name: HisLink Spin Column]. Thereafter, the resin in the column was washed to remove unbound proteins and the like. Next, the protein adsorbed on the column was eluted with 0.2 mL of an elution buffer (composition, 100 mM hepes-sodium hydroxide buffer (pH 7.5), 500 mM imidazole), and a fraction containing WT was collected. .
  • an elution buffer composition, 100 mM hepes-sodium hydroxide buffer (pH 7.5), 500 mM imidazole
  • the magnitude of the effective charge in the DNA interaction region of WT is as follows: the charge score of each of lysine residues and arginine residues which are positively charged amino acid residues is “+1”, and the aspartic acid residue which is a negatively charged amino acid residue When the charge score of each of the glutamic acid residues is “ ⁇ 1” and calculated according to the above formula (I), it is +7.
  • FIG. 1 shows the localization positions of amino acid residues to be substituted selected in Production Example 2 in wild-type MMLV reverse transcriptase.
  • the site-specific mutagenesis primer is a primer designed to cause substitution of amino acid residues shown in Table 1.
  • Test Example 1 In the incubation solution [composition: 10 mM potassium phosphate buffer (pH 7.6), 2 mM dithiothreitol, 0.2 vol% Triton TM X-100 and 10 vol% glycerol], the WT obtained in Production Example 1 ( 100 nM) or the single mutant (100 nM) obtained in Production Example 2 was subjected to heat treatment by incubation at 50 ° C. for 15 minutes in the presence or absence of 28 ⁇ M poly (rA) ⁇ p (dT) 15 . The WT or the single mutant was then incubated for 30-60 minutes on ice.
  • reaction solution [Composition: 25 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 2 mM dithiothreitol, 5 mM magnesium chloride, 12.5 ⁇ M poly (rA) ⁇ p (dT) 15 [p (dT) 15 concentration in terms], 0.2 mM - were [methyl 3 H] in dTTP (1.85Bq / pmol) [GE Healthcare (GE Healthcare) Co. Ltd.]], the 10 nM WT or alone variant were incubated at 37 ° C..
  • the initial reaction rate was calculated based on the change over time in the dTTP uptake amount.
  • the residual activity was calculated from the initial reaction rate when the heat treatment was not performed (referred to as “initial reaction rate A”) and the initial reaction rate when the heat treatment was performed (referred to as “initial reaction rate B”). .
  • the residual activity is represented by the formula (II):
  • Residual activity (initial reaction rate B / initial reaction rate A) ⁇ 100 (II)
  • the amino acid residue to be substituted provides higher thermal stability than WT by introducing site-specific mutations. Whether it was a residue was evaluated.
  • the evaluation criteria are shown in Table 2, and the evaluation results are shown in Table 3.
  • the results of examining the relationship between the type of amino acid residue substitution and the residual activity in Test Example 1 are shown in FIG.
  • FIG. 2 shows the residual activity of representative examples of WT and single mutants.
  • results shown in FIG. 2 indicate that the residual activity of each of the two single mutants exceeds 15% among the three types of single mutants in which site-specific mutations are introduced into E286. Moreover, it turns out that the residual activity of each of two types of single mutants exceeds 15% among three types of single mutants in which site-specific mutations are introduced into D124.
  • Example 1 From the substitution target amino acid residues evaluated in Test Example 1, the substitution target amino acid residue having an evaluation of AA was selected. Next, four types of mutants (E302K, L435R, D124R, and E286R) were selected from the mutants in which the selected amino acid residue was substituted with another amino acid residue in descending order of residual activity.
  • Production Example 2 a primer designed to cause substitution of four selected amino acid residues was used in place of the primer designed to cause substitution of amino acid residues shown in Table 1. Except for, the same operation as in Production Example 2 was performed to obtain a multiple mutant of MMLV reverse transcriptase (D124R / E286R / E302K / L435R). As a result of SDS-PAGE, the obtained multiple mutant was confirmed to show a single band of 75 kDa.
  • the charge scores of lysine residues and arginine residues that are positively charged amino acid residues are “+1”, the charge scores of aspartic acid residues and glutamic acid residues that are negatively charged amino acid residues are “ ⁇ 1”, and DNA Based on the number of lysine residues, arginine residues, aspartic acid residues and glutamic acid residues in the interaction region, the magnitude of the effective charge in the DNA interaction region of the multiple mutant is calculated using the above formula (I). The score was calculated. As a result, the score of the magnitude of the effective charge in the DNA interaction region of the obtained multiple mutant was +13.
  • Example 2 In Example 1, instead of the primers designed to cause substitution of the four selected amino acid residues, substitution of the four amino acid residues selected in Example 1 and 524 in SEQ ID NO: 2
  • the MMLV reverse transcriptase multiple mutant (D124R / E286R / D) was carried out in the same manner as in Example 1 except that a primer designed so that substitution of Asp to Ala at position (D524A) was used. E302K / L435R / D524A).
  • Asp at position 524 in SEQ ID NO: 2 is an amino acid residue that is located in the active site of the WT RNase H reaction and is essential for catalytic activity.
  • SDS-PAGE analysis the obtained multiple mutant was confirmed to show a single band of 75 kDa.
  • the score of the magnitude of the effective charge in the DNA interaction region of the obtained multiple mutant was +13.
  • Example 3 Among the four types of amino acid residue substitutions selected in Example 1, substitution of three types of amino acid residues E302K, L435R and E286R was selected. Next, in Example 1, a primer designed to cause substitution of the three types of amino acid residues is used in place of the primer designed to cause substitution of the selected four types of amino acid residues. Except for the above, the same operation as in Example 1 was performed to obtain a multiple mutant of MMLV reverse transcriptase (E286R / E302K / L435R). As a result of SDS-PAGE analysis, the obtained multiple mutant was confirmed to show a single band of 75 kDa.
  • the score of the magnitude of the effective charge in the DNA interaction region of the obtained multiple mutant was +11.
  • Example 4 In Example 1, in place of the primers designed to cause substitution of the four selected amino acid residues, the substitution of the three amino acid residues selected in Example 3 and D524A occurs. Except that the designed primer was used, the same operation as in Example 1 was performed to obtain a multiple mutant of MMLV reverse transcriptase (E286R / E302K / L435R / D524A). As a result of SDS-PAGE analysis, the obtained multiple mutant was confirmed to show a single band of 75 kDa.
  • the score of the magnitude of the effective charge in the DNA interaction region of the obtained multiple mutant was +11.
  • Test Example 2 In Test Example 1, the same procedure as in Test Example 1 was performed, except that the multiple mutants obtained in Examples 1 to 4 were used instead of the single mutant obtained in Production Example 2, and the remaining activity was Was calculated.
  • the results of examining the relationship between the type of multiple mutant and the residual activity in Test Example 2 are shown in FIG.
  • 1 is the residual activity of the multiple mutant obtained in Example 1
  • 2 is the residual activity of the multiple mutant obtained in Example 2
  • 3 is the residual activity of the multiple mutant obtained in Example 3.
  • 4 shows the residual activity of the multiple mutant obtained in Example 4.
  • the white bar indicates the residual activity of the multiple mutant in the absence of the template primer
  • the black bar indicates the residual activity of the multiple mutant in the presence of the template primer.
  • the effective charge magnitude score of the region of the multiple mutant obtained in each of Examples 1 to 4 is +11 to +13, and from the effective charge magnitude score (+7) of the WT region. Is also big. Therefore, from this result, amino acid residues in the DNA interaction region are determined to be positively charged amino acids so that the effective charge magnitude score of the DNA interaction region is larger than the effective charge in the WT DNA interaction region. It can be seen that high thermal stability can be ensured by substituting a residue or a non-polar amino acid residue and localizing a positively charged amino acid residue or a non-polar amino acid residue in the DNA interaction region.
  • Test Example 3 Reaction solution [Composition: 25 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 2 mM dithiothreitol, 5 mM magnesium chloride, poly (rA) ⁇ p (dT) 15 [p ( dT) 15 in terms of concentration], 0.2 mM [methyl - 3 H] dTTP (1.85Bq / pmol) [in GE Healthcare (GE Healthcare) Co. Ltd.]], multiple mutants obtained in example 3, performed Multiple mutants obtained in Example 4 or WT (Comparative Example 1) (5 nM) were incubated at 37 ° C.
  • the glass filter was dried.
  • the glass filter was placed in 2.5 mL of a liquid scintillation reagent (trade name: Ecoscint H, manufactured by National Diagnostics), and the radioactivity was counted. Based on the radioactivity, dTTP uptake was calculated. Based on the change over time in the amount of dTTP uptake, the initial reaction rate was calculated.
  • a liquid scintillation reagent trade name: Ecoscint H, manufactured by National Diagnostics
  • the k cat / K m values of the multiple mutants obtained in Example 3 and the multiple mutants obtained in Example 4 are the WT k cat / K m values. It can be seen that they are 170% and 130%. From these results, it is suggested that the catalytic efficiency of the multiple mutant obtained in Example 3 and the multiple mutant obtained in Example 4 is higher than the catalytic efficiency of WT.
  • Test Example 4 Multiple mutations obtained in Example 3 in incubation solution [composition: 10 mM potassium phosphate buffer (pH 7.6), 2 mM dithiothreitol, 0.2 vol% Triton TM X-100, 10 vol% glycerol] Or the multiple mutant (100 nM) obtained in Example 4 in the presence of 28 ⁇ M poly (rA) ⁇ p (dT) 15 at 52-58 ° C. for a certain time (1, 2, 5, 10 or 15 minutes) Incubation was followed by heat treatment, followed by incubation on ice for 30-60 minutes.
  • WT Comparative Example 1
  • WT was incubated at 48 to 52 ° C. for a predetermined time (1, 2, 5, 10, or 15 minutes) in the presence of 28 ⁇ M poly (rA) ⁇ p (dT) 15. Then, heat treatment was performed, followed by incubation for 30 to 60 minutes on ice.
  • reaction solution [Composition: 25 mM Tris-HCl buffer (pH 8.3), 50 mM potassium chloride, 2 mM dithiothreitol, 5 mM magnesium chloride, 12.5 ⁇ M poly (rA) ⁇ p (dT) 15 [p (dT) 15 equivalent concentration], 0.2 mM [methyl- 3 H] dTTP (1.85 Bq / pmol) [manufactured by GE Healthcare]], multiple mutant obtained in Example 3, Example 4 Multiple mutants obtained in the above or WT (comparative example) (10 nM) were incubated at 37 ° C.
  • the glass filter was dried.
  • the glass filter was placed in 2.5 mL of a liquid scintillation reagent (trade name: Ecoscint H, manufactured by National Diagnostics), and the radioactivity was counted. Based on the radioactivity, dTTP uptake was calculated.
  • a liquid scintillation reagent trade name: Ecoscint H, manufactured by National Diagnostics
  • the initial reaction rate was calculated based on the change over time in the dTTP uptake amount.
  • the residual activity was calculated from the initial reaction rate when the heat treatment was not performed (referred to as “initial reaction rate a”) and the initial reaction rate when the heat treatment was performed (referred to as “initial reaction rate b”).
  • the residual activity is represented by the formula (III):
  • Residual activity (initial reaction rate b / initial reaction rate a) ⁇ 100 (III)
  • Test Example 4 the results of examining the relationship between the incubation time and ln [residual activity (%)] are shown in FIG. FIG. 4 shows the results when heat treatment is performed at 52.degree.
  • ln [residual activity (%)] represents a natural value of the residual activity.
  • the initial activity of each of the multiple mutants obtained in Example 3, the multiple mutants obtained in Example 4 and WT is reduced to 50% after 10 minutes incubation.
  • the temperature T 50 required for the calculation was calculated.
  • the temperature T 50 required to reduce the initial activity of each of the multiple mutants obtained in Example 3, the multiple mutants obtained in Example 4 and WT to 50% after 10 minutes incubation is Were estimated to be 45.1, 54.2 and 55.9 ° C., respectively.
  • the activation energy (E a ) of thermal inactivation of the multiple mutant obtained in Example 3, the multiple mutant obtained in Example 4, and WT, respectively. was calculated.
  • the thermal inactivation activation energies (E a ) of the multiple mutant obtained in Example 3, the multiple mutant obtained in Example 4 and the WT were 240, 298 and 322 kJ / mol, respectively. It was estimated that. From these results, it can be seen that the multiple mutant obtained in Example 3 and the multiple mutant obtained in Example 4 are more stable than WT.
  • coli RNA solution (1.0 ⁇ g / ⁇ L), multiple mutants obtained in Example 3, in Example 4 Enzyme solution of the obtained multiple mutant or WT (Comparative Example 1) [Composition of solvent used in enzyme solution: 10 mM potassium phosphate buffer (pH 7.6), 2 mM dithiothreitol, 0.2 vol% Triton TM X-100, 10 volume% glycerol] 2 ⁇ L was added and mixed to prepare 20 ⁇ L of the reaction mixture.
  • RNA 1014 nucleotide RNA corresponding to the DNA sequence 8353-9366 of the cesA gene (Genbank accession number: DQ360825) of Bacillus cereus was prepared by in vitro transcription. Table 5 shows the base sequence and SEQ ID NO of the RV-R26 primer.
  • the obtained reaction mixture was incubated at 46 to 64 ° C. for 30 minutes, and then heated at 95 ° C. for 5 minutes.
  • 3 ⁇ L of the obtained product 18 ⁇ L of water, 10 ⁇ PCR buffer (composition: 500 mM potassium chloride, 100 mM Tris-HCl buffer (pH 8.3), 15 mM magnesium chloride) 3 ⁇ L, 10 ⁇ M F5 primer aqueous solution 1 ⁇ L 1 ⁇ L of 10 ⁇ M RV primer aqueous solution, 3 ⁇ L of 2.0 mM dNTP mixture, and 1 ⁇ L of recombinant Taq polymerase solution (manufactured by Toyobo Co., Ltd., 1 U / ⁇ L) were mixed to prepare 30 ⁇ L of a PCR mixture.
  • 10 ⁇ PCR buffer composition: 500 mM potassium chloride, 100 mM Tris-HCl buffer (pH 8.3), 15 mM magnesium chloride
  • PCR was performed using the obtained PCR mixture. PCR was performed by carrying out 30 cycles of reaction at 95 ° C. for 30 seconds, followed by 30 cycles of 95 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds.
  • the base sequences and SEQ ID NOs of the F5 primer and RV primer are as shown in Table 5.
  • the obtained amplification products were separated by electrophoresis using a 1.0% by mass agarose gel and stained with ethidium bromide (1 ⁇ g / mL).
  • the results of electrophoresis in Test Example 5 are shown in FIG.
  • the highest temperature at which the multiple mutant obtained in Example 3 and the multiple mutant obtained in Example 4 exhibit cDNA synthesis activity is 60 ° C.
  • WT Comparative Example
  • the highest temperature at which 1) exhibits cDNA synthesis activity is 54 ° C. From these results, it can be seen that the multiple mutant obtained in Example 3 and the multiple mutant obtained in Example 4 show cDNA synthesis activity at a temperature higher than that of WT.
  • Example 5 Among the mutants in which the amino acid residue to be substituted, which is evaluated as AA in Test Example 1, is substituted with another amino acid residue, except for E302K, the three mutants (L435R, D124R) in descending order of remaining activity. And E286R) were selected.
  • Production Example 2 a primer designed to cause substitution of three selected amino acid residues was used in place of the primer designed to cause substitution of amino acid residues shown in Table 1. Except for the above, the same operation as in Production Example 2 was performed to obtain a multiple mutant of MMLV reverse transcriptase (D124R / E286R / L435R). As a result of SDS-PAGE analysis, the obtained multiple mutants were confirmed to show a single band of 75 kDa.
  • the score of the magnitude of the effective charge in the DNA interaction region of the obtained multiple mutant was +11.
  • Example 6 As a site-specific mutagenesis primer, it was designed to cause substitution of three selected amino acid residues and substitution of Asp at position 524 to Ala (D524A) in SEQ ID NO: 2. The same procedure as in Example 5 was performed except that the primers were used, and a multiple mutant of MMLV reverse transcriptase (D124R / E286R / L435R / D524A) was obtained. As a result of SDS-PAGE analysis, the obtained multiple mutant was confirmed to show a single band of 75 kDa.
  • the score of the magnitude of the effective charge in the DNA interaction region of the obtained multiple mutant was +11.
  • Example 7 In Example 5, the same operation as in Example 5 was performed, except that a primer designed to cause substitution of two types of amino acid residues, L435R and E286R, was used as a site-specific mutation primer. Multiple mutants of MMLV reverse transcriptase (E286R / L435R) were obtained. As a result of SDS-PAGE analysis, the obtained multiple mutant was confirmed to show a single band of 75 kDa.
  • Example 8 In Example 5, as the primer for site-directed mutagenesis, except that a primer designed to generate substitution of two kinds of amino acid residues selected in Example 7 and D524A was used. The same operation was performed to obtain a multiple mutant of MMLV reverse transcriptase (E286R / L435R / D524A). As a result of SDS-PAGE analysis, the obtained multiple mutant was confirmed to show a single band of 75 kDa.
  • Test Example 6 In Test Example 1, the same procedure as in Test Example 1 was carried out except that the multiple mutants obtained in Examples 5 to 8 were used instead of the single mutant obtained in Production Example 2. Was calculated.
  • FIG. 7 shows the results of examining the relationship between the type of multiple mutant and the residual activity of the multiple mutant in the presence of the template primer in Test Example 6. In the figure, 1 is the residual activity of the multiple mutant obtained in Example 5, 2 is the residual activity of the multiple mutant obtained in Example 6, and 3 is the residual activity of the multiple mutant obtained in Example 7. 4 shows the residual activity of the multiple mutant obtained in Example 8.
  • a region related to the interaction with the template primer in wild type MMLV reverse transcriptase (corresponding to the threonine residue at position 24 to the proline residue at position 474 in the amino acid sequence shown in SEQ ID NO: 2). It is understood that a mutant reverse transcriptase having high thermostability can be obtained by substituting at least E286 with a positively charged amino acid residue or a nonpolar amino acid residue in the region).
  • amino acid residues in the DNA interaction region are defined as positively charged amino acid residues or non-charged amino acid residues such that the effective charge magnitude score of the DNA interaction region is greater than the net charge in the WT DNA interaction region. It can be seen that high thermal stability can be ensured by substituting with polar amino acid residues and localizing positively charged amino acid residues or nonpolar amino acid residues in the DNA interaction region.
  • mutant reverse transcriptase (mutant reverse transcriptase of the present invention) has a high thermostability, and therefore has a high reverse transcriptase activity even when used for a reaction at a high reaction temperature. Is expressed. Therefore, according to the mutant reverse transcriptase of the present invention, even when the RNA used as the template contains a sequence that easily forms a secondary structure, the reaction temperature during the reverse transcription reaction is set to a high temperature. Thus, formation of secondary structure can be suppressed and reverse transcription can be performed.
  • the mutant reverse transcriptase of the present invention is not limited to the RNA-containing sample to be used and is a highly versatile analytical reagent (for example, reverse transcription reaction kit), a reagent for detecting viruses, bacteria, diseases, etc. It is suggested to be useful as (for example, a detection kit).
  • a highly versatile analytical reagent for example, reverse transcription reaction kit
  • nucleic acid-related enzymes including reverse transcriptase have a nucleic acid interaction region that interacts with nucleic acid (a DNA interaction region in reverse transcriptase). Therefore, as in the case of the mutant reverse transcriptase of the present invention, high thermal stability is ensured by substituting amino acid residues in the nucleic acid interaction region with positively charged amino acid residues or nonpolar amino acid residues. Expected to be able to.
  • Example 1-10x reverse transcriptase buffer [Composition: 250 mM Tris-HCl buffer (pH 8.3), 500 mM potassium chloride, 20 mM dithiothreitol] -2.0 mM dNTP mixture-10 ⁇ M primer aqueous solution-Standard RNA solution (1.6 pg / ⁇ L)
  • Example 1-10x reverse transcriptase buffer [Composition: 250 mM Tris-HCl buffer (pH 8.3), 500 mM potassium chloride, 20 mM dithiothreitol] -2.0 mM dNTP mixture-10 ⁇ M RV-R26 primer aqueous solution-Standard RNA solution (1.6 pg / ⁇ L) -E. coli RNA solution (1.0 ⁇ g / ⁇ L)
  • SEQ ID NO: 3 is the sequence of the RV-R26 primer.
  • SEQ ID NO: 4 is the sequence of F5 primer.
  • SEQ ID NO: 5 is the sequence of the RV primer.

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Abstract

L'invention concerne une transcriptase inverse variante qui présente une stabilité à la chaleur élevée et une polyvalence élevée ; un acide nucléique de celle-ci ; un procédé de production de la transcriptase inverse variante ; un kit de transcription inverse très polyvalent ; un kit de détection très polyvalent ; un procédé pour améliorer la stabilité à la chaleur d'une enzyme pour acide nucléique grâce auquel la stabilité à la chaleur de l'enzyme pour acide nucléique peut être considérablement améliorée ; et un procédé de transcription inverse grâce auquel une transcription inverse peut être efficacement effectuée. Un résidu d'acide aminé dans un domaine d'interaction avec l'acide nucléique d'une enzyme sauvage est remplacé par un résidu d'acide aminé positivement chargé ou un résidu d'acide aminé non polaire pour former un domaine d'interaction avec l'acide nucléique présentant une charge nette positive plus élevée que celle du domaine d'interaction avec l'acide nucléique de l'enzyme sauvage.
PCT/JP2011/068157 2010-08-13 2011-08-09 Transcriptase inverse variante WO2012020759A1 (fr)

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JP2014082936A (ja) * 2012-10-19 2014-05-12 Kyoto Univ 変異型逆転写酵素
JP2014124166A (ja) * 2012-12-27 2014-07-07 Tosoh Corp 酵素のスクリーニング方法
WO2015075842A1 (fr) * 2013-11-25 2015-05-28 国立大学法人京都大学 Transcriptase inverse variante
EP2998394A4 (fr) * 2013-05-17 2016-12-28 Consejo Superior De Investig Cientificas (Csic) Rétrotranscriptases du vih type 1 groupe o, actives à températures élevées
JP2017104092A (ja) * 2015-11-27 2017-06-15 国立大学法人京都大学 新規核酸合成法
JP2017131164A (ja) * 2016-01-28 2017-08-03 東洋紡株式会社 改良されたウイルス検出方法
WO2018110595A1 (fr) * 2016-12-14 2018-06-21 タカラバイオ株式会社 Variant de transcriptase inverse résistant à la chaleur
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EP3848458A4 (fr) * 2019-11-13 2022-11-30 Daan Gene Co., Ltd. Mutant de transcriptase inverse thermostable et son utilisation

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN106414704B (zh) 2014-03-14 2020-11-03 生命技术公司 用于核酸扩增和检测的集成***
AU2015298571B2 (en) 2014-07-30 2020-09-03 President And Fellows Of Harvard College Cas9 proteins including ligand-dependent inteins
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JP2020510439A (ja) 2017-03-10 2020-04-09 プレジデント アンド フェローズ オブ ハーバード カレッジ シトシンからグアニンへの塩基編集因子
SG11201908658TA (en) 2017-03-23 2019-10-30 Harvard College Nucleobase editors comprising nucleic acid programmable dna binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
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DE102018214123A1 (de) * 2018-08-21 2020-02-27 Gühring KG Zerspanungswerkzeug
CN113785053B (zh) * 2018-12-26 2024-07-12 深圳华大生命科学研究院 酶活性提高的逆转录酶及其应用
DE112020001342T5 (de) 2019-03-19 2022-01-13 President and Fellows of Harvard College Verfahren und Zusammensetzungen zum Editing von Nukleotidsequenzen
CN114174503B (zh) * 2019-07-26 2024-06-28 东洋纺株式会社 稳定性优异的突变型逆转录酶
EP4146804A1 (fr) 2020-05-08 2023-03-15 The Broad Institute Inc. Méthodes et compositions d'édition simultanée des deux brins d'une séquence nucléotidique double brin cible

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004511211A (ja) 2000-05-26 2004-04-15 インヴィトロジェン　コーポレーション 耐熱性逆転写酵素およびその使用
JP2009504162A (ja) * 2005-08-10 2009-02-05 ストラタジーン カリフォルニア 変異体逆転写酵素および使用方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9783791B2 (en) * 2005-08-10 2017-10-10 Agilent Technologies, Inc. Mutant reverse transcriptase and methods of use
GB0806562D0 (en) 2008-04-10 2008-05-14 Fermentas Uab Production of nucleic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004511211A (ja) 2000-05-26 2004-04-15 インヴィトロジェン　コーポレーション 耐熱性逆転写酵素およびその使用
JP2009504162A (ja) * 2005-08-10 2009-02-05 ストラタジーン カリフォルニア 変異体逆転写酵素および使用方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AREZI B. ET AL.: "Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer", NUCLEIC ACIDS RES., vol. 37, no. 2, 2009, pages 473 - 481, XP002556110 *
BAHRAM AREZI ET AL.: "Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer", NUCLEIC ACIDS RESEARCH, vol. 37, 4 December 2008 (2008-12-04), pages 473 - 481
MIZUNO M. ET AL.: "Insight into the mechanism of the stabilization of moloney murine leukaemia virus reverse transcriptase by eliminating RNase H activity", BIOSCI.BIOTECHNOL. BIOCHEM., vol. 74, no. 2, 7 February 2010 (2010-02-07), pages 440 - 442, XP055075589 *
See also references of EP2604688A4 *
YASUKAWA K. ET AL.: "Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis", J.BIOTECHNOL., vol. 150, no. 3, 8 October 2010 (2010-10-08), pages 299 - 306, XP027483996 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014082936A (ja) * 2012-10-19 2014-05-12 Kyoto Univ 変異型逆転写酵素
JP2014124166A (ja) * 2012-12-27 2014-07-07 Tosoh Corp 酵素のスクリーニング方法
EP2998394A4 (fr) * 2013-05-17 2016-12-28 Consejo Superior De Investig Cientificas (Csic) Rétrotranscriptases du vih type 1 groupe o, actives à températures élevées
WO2015075842A1 (fr) * 2013-11-25 2015-05-28 国立大学法人京都大学 Transcriptase inverse variante
JP2017104092A (ja) * 2015-11-27 2017-06-15 国立大学法人京都大学 新規核酸合成法
JP2017131164A (ja) * 2016-01-28 2017-08-03 東洋紡株式会社 改良されたウイルス検出方法
WO2018110595A1 (fr) * 2016-12-14 2018-06-21 タカラバイオ株式会社 Variant de transcriptase inverse résistant à la chaleur
JPWO2018110595A1 (ja) * 2016-12-14 2019-10-24 タカラバイオ株式会社 耐熱性の逆転写酵素変異体
US11220677B2 (en) 2016-12-14 2022-01-11 Takara Bio Inc. Heat-resistant reverse transcriptase mutant
JP7061078B2 (ja) 2016-12-14 2022-04-27 タカラバイオ株式会社 耐熱性の逆転写酵素変異体
WO2018198682A1 (fr) * 2017-04-26 2018-11-01 東洋紡株式会社 Procédé d'analyse de virus et kit d'analyse de virus
JPWO2018198682A1 (ja) * 2017-04-26 2020-03-12 東洋紡株式会社 ウイルスの検査方法およびウイルスの検査用キット
JP7167913B2 (ja) 2017-04-26 2022-11-09 東洋紡株式会社 ウイルスの検査方法およびウイルスの検査用キット
EP3848458A4 (fr) * 2019-11-13 2022-11-30 Daan Gene Co., Ltd. Mutant de transcriptase inverse thermostable et son utilisation

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US8900814B2 (en) 2014-12-02
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JP2016136970A (ja) 2016-08-04
EP2604688B1 (fr) 2018-01-10
EP2604688A1 (fr) 2013-06-19
US20130143225A1 (en) 2013-06-06

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